In many applications it is desirable to enrich, or alternatively deplete, certain cell populations in a biological sample. The fields of hematology, immunology and oncology rely on samples of peripheral blood and cell suspensions from related tissues such as bone marrow, spleen, thymus and fetal liver. The separation of specific cell types from these heterogeneous samples is key to research in these fields. Purified populations of immune cells such as T cells and B cells are necessary for the study of immune function and are used in immunotherapy. Investigation of the cellular, molecular and biochemical processes require analysis of certain cell types in isolation. Numerous techniques have been used to isolate or deplete erythrocytes, lymphocyte subsets such as T cells, B cells and natural killer (NK) cells and granulocytes such as neutrophils, basophils and eosinophils.
Hematopoietic cells and immune cells have been separated on the basis of physical characteristics such as density and through direct targeting with monoclonal antibodies and a solid surface such as magnetic particles. There are two basic approaches to separating cell populations from peripheral blood and related cell suspensions using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labelled with the antibody(s). In positive selection techniques, the desired cells are labelled with antibodies and removed from the remaining unlabelled/undesired cells. In negative selection, the undesired cells are labelled and removed. Antibody and complement treatment and the use of immunotoxins is a negative selection technique, whereas fluorescence assisted cell sorting (FACS) and most bulk immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques, cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e.g. column of beads, flasks, non-magnetic and magnetic particles. Immunoadsorption techniques have won favour clinically and in research because they maintain the high specificity of targeting cells with monoclonal antibodies, but unlike FACS, they can be scaled up to deal directly with the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins and complement.
Magnetic separation is a process used to selectively retain magnetic materials within a vessel, such as a centrifuge tube or column, in a magnetic field gradient. Targets of interest, such as specific biological cells, proteins and nucleic acids, can be magnetically labeled by binding of magnetic particles to the surface of the targets through specific interactions including immuno-affinity interactions. Other useful interactions include drug-drug receptor, antibody-antigen, hormone-hormone receptor, growth factor-growth factor receptor, carbohydrate-lectin, nucleic acid sequence-complementary nucleic acid sequence, enzyme-cofactor or enzyme-inhibitor binding. The suspension, containing the targets of interest within a suitable vessel, is then exposed to magnetic field gradients of sufficient strength to separate the targets from other entities in the suspension. The vessel can then be washed with a suitable fluid to remove the unlabeled entities, resulting in a purified suspension of the targets of interest.
The advent of monoclonal antibodies against cell surface antigens has greatly expanded the potential to distinguish and separate distinct cell types. The majority of magnetic labeling systems use supramagnetic particles with monoclonal antibodies or streptavidin covalently bound to their surface. In cell separation applications these particles can be used for either positive selection, where the desired cells are magnetically labeled, or negative selection where the majority of undesired cells are magnetically labeled. Magnetic separation applications where the targets of interest are proteins or nucleic acids would be considered positive selection approaches since the target entity of interest is typically captured on the magnetic particle.
Several commercial cell separation products are available that utilize a magnetic particle directly coupled to antibodies (Miltenyi Biotec Inc., Gladbach, Germany, Life Technologies Corp., Carlsbad, USA, BD Biosciences, San Jose, USA.). Other approaches utilize the labelling of target cells with specific antibodies conjugated to biotin followed by the addition of streptavidin coated magnetic particles that bind the biotinylated antibodies (Miltenyi Biotec, Inc. Life Technologies, BD Biosciences, and STEMCELL Technologies Inc., Vancouver, Canada). Another example is the EasySep™ cell separation system (STEMCELL Technologies Inc.) whereby a bi-specific tetrameric antibody complex is used to crosslink magnetic particles to cells of interest. Tetrameric antibody complexes (TAC) are comprised of two monoclonal antibodies from a first species held in tetrameric array by two antibodies from a second species that bind to the Fc-fragment of the antibodies from the first species (See U.S. Pat. No. 4,868,109 to Lansdorp, which is incorporated herein by reference for a description of TACs and methods for preparing the same).
In the preparation of bi-specific TACs, three different TACs can be formed which comprise the final antibody composition. If an equivalent concentration of two different antibodies (A and B) from the first animal species are combined with an equimolar amount of the crosslinking antibody from the second animal species (C), 25% will be mono-specific TAC for antibody A, 25% will be mono-specific TAC for the second antibody B, and 50% of the TAC will be bi-specific for antibodies A and B. The ratio of each antibody from the first species can be manipulated to skew the ratio of mono-specific to bi-specific TACs to the first or second antibody specific for their respective target antigens.
As used in the current invention, a mono-specific TAC is specific for a single target entity. In one embodiment, the mono-specific TAC contains two identical antibodies from the first animal species that recognize the same antigenic epitope held in a tetrameric array by two antibodies from a second animal species that recognize the Fc-fragment of the first animal species. In another embodiment, the mono-specific TAC contains two different antibody clones from the first animal species that recognize different epitopes on the same target antigen that are held in a tetrameric array by two antibodies from a second animal species that recognize the Fc-fragment of the first animal species. In yet another embodiment, the mono-specific TAC contains two different antibody clones from the first animal species that recognize different antigens expressed on the same target entity that are held in a tetrameric array by two antibodies from a second animal species that recognize the Fc-fragment of the first animal species.
The mono-specific TAC can increase the valency of the complex for its target entity as the TAC would have four antigen binding sites compared to just two with a single IgG antibody molecule.
Patents describing antibodies directly coupled to particles either directly or indirectly via an intermediate receptor-ligand interaction whereby either one of the receptor or ligand are first coupled to the magnetic particle have been described in U.S. Pat. No. 3,970,518A, U.S. Pat. No. 4,230,685, U.S. Pat. No. 8,298,782B2, U.S. Pat. No. 7,160,723B2, and U.S. Pat. No. 5,543,289A which are incorporated herein by reference. In each of these examples, either single or multiple antibodies that recognize a target entity are coupled to particles using conventional techniques that are readily apparent to those skilled in the art such as physical adsorption or chemical conjugation.
Physical adsorption of ligands such as antibodies onto solid surfaces plays a critical role in numerous natural processes and holds great utility in biomaterial applications. Despite efforts and progress in understanding protein adsorption phenomenon at solid surfaces there is widely differing and contradictive explanations as to the observed phenomena that occurs when a protein adsorbs onto a solid surface such as a flask, column of beads, or particles [1]. Protein adsorption onto solid surfaces can be affected by a numerous factors including the pH and ionic strength of the reaction buffer, the temperature of the reaction and the isoelectric point of the protein. In addition, the size, surface charge, reactive moieties on the surface also contribute greatly to the adsorption of the proteins. Once the protein is initially adsorbed or concentrated on the surface, it can be covalently conjugated to said surface.
Depending on the adsorption conditions or conjugation chemistry, the orientation of the antibody can be bound in a conformation that does not allow it to functionally bind to its target antigen. The orientation of the antibodies can be manipulated by modifying the adsorption or conjugation reaction conditions or by the use of an intermediate that is first conjugated to a surface that can help to orient the antibody with the reactive Fab binding domains oriented outwards from the surface (see U.S. Pat. No. 8,298,782 B2 or U.S. Pat. No. 4,230,685). Even in the case of the coupling of an antibody binding intermediate such as protein A or streptavidin, the coupling of said intermediate can also be inefficient resulting in less than ideal coupling efficiency.
In view of the foregoing, there is a need in the art to provide simple and novel methods for improving antibody coupling methods for the preparation of surfaces specific for a target entity for use in fractionating mixtures of target entities and non-target entities.